Project Summary/Abstract Our genomic DNA encodes critical information that is required for the healthy function of every cell, tissue, and organ. However, DNA is continuously accumulating toxic damage that arises during normal cellular processes, or is caused by environmental conditions such as sunlight and chemical carcinogens. Double-stranded DNA breaks (DSBs) are the most dangerous lesions. DSBs occur when both strands of the DNA double helix are broken in close proximity to each other, fragmenting the chromosome into two distinct pieces. If unrepaired, even a single DSB can initiate cellular dysfunction, malignant transformation, and tumor growth. Our cells can repair DSBs via two distinct pathways: a rapid, error-prone reaction or via a second process that is largely error-free. Remarkably, the primary molecular steps that determine the DNA repair pathway are still not completely known. Thus, there is a critical need to understand how healthy cells repair their fragmented DNA and how disruptions in these processes can lead to cancer. My long-tern goal is to understand how specialized DNA repair proteins serve as the molecular caretakers of the genome. In my graduate work, I will investigate how a group of human enzymes coordinate the first steps of DSB repair. I will first investigate how the Mre11/Rad50/Nbs1 (MRN) complex acts as the molecular sensor for DSBs. I will also explore how MRN harnesses its multiple biochemical activities to begin processing the free DNA ends. Next, I will determine how MRN recruits additional enzymes, and how this spatially and temporally ordered assembly of these proteins catalyzes the first biochemical steps that determine the DSB repair pathway. As I transition into a postdoctoral position, I will characterize how these DNA repair proteins recognize and are blocked at the normal ends of DNA, telomeres. Deciphering these critical molecular events remains challenging because traditional approaches are unable to directly observe the intricate molecular choreography of multiple repair proteins on the same DNA molecule. To achieve my aims, I have pioneered a unique, ultra-sensitive microscopy technique that can image individual molecules of DNA and record movies of multiple enzymes as they repair DNA in real time. Using this fluorescence microscope, I will directly observe how critical human enzymes coordinate their actions to initiate error-free DNA repair. The anticipated results of these studies will answer a long-standing question of how human DNA is repaired. Ultimately, this knowledge will be required for developing new diagnostics and therapeutics that specifically target cancer cells that have lost the ability to correctly repair their genomes.